The conjugation of proteins with ubiquitin involves the sequential action of proteins known as E1 (ubiquitin activating enzyme), E2s or UBCs (ubiquitin conjugating enzymes), and E3s (ubiquitin protein ligases). We have previously determined that RING finger proteins are, in general, ubiquitin ligases and are now focused on understanding issues related to the mechanism of action and substrates of specific members of this family as well as identifying inhibitors. Our studies looking for inhibitors of Hdm2 have resulted in the identification of a family of compounds that inhibit Hdm2 E3 activity both in vitro and in cells (HLI98 family) and we have now identified a related small molecule that is highly soluble in water and exhibits greater potency than the HLI98 compounds. This small molecule, HLI373, selectively kills p53-expressing cancer cells in a manner similar to the original compounds. Additionally, our screens have also resulted in the identification of an inhibitor of the ubiquitin E1 inhibitor. This inhibitor markedly deceases ubiquitin-mediated degradation of substrates as well as non-proteolytic functions of the ubiquitin system including ligand-induced activation of NfkB and ligand-mediated ubiquitination of the epidermal growth factor receptor. It also results in increased levels of p53 and in selective cell death in transformed cells and may also serve as the basis for new therapeutics for cancer and other diseases in which perturbing functions of the ubiquitin system may be of beneficial. In this regard, the NCI has recently decided to initiate developmental studies aimed towards assessing the potential for both our Hdm2 and E1 inhibitors to serve as the basis for the development of novel cancer therapeutics. We are also carrying out structure-function and structural studies on the transmembrane prometastatic endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase gp78. We had previously identified a second E2 binding site on gp78 specific for the E2 Ube2g2, known as the Ube2g2 Binding Region (G2BR). We have now determined that the G2BR binds to a region of Ube2g2 distinct from where the RING finger binds this E2 and increases the affinity of Ube2g2 for the gp78 RING finger and therefore stimulates ubiquitination. We predict that this is likely to be the first of many such binding sites on the backside of E2s and that these types of interaction may represent a general means by which the affinity of E2s for RING finger E3s is increased. This research, which involved a combination of X-ray crystallography, NMR and biochemistry, was recently published in Molecular Cell and was accompanied by an NCI press release as well as a Preview in the journal Structure, that specifically highlighted our findings. Continued evaluation of the means by which gp78 functions as an E3 and development of possible inhibitors is ongoing. Additionally, we are evaluating other specific RING finger E3s for additional sites of E2 bindings that might influence function. In addition to this we have been carrying out structure-function studies for the HRD1 ubiquitin ligase of yeast. In particular we have been focusing on an essential component of this ligase complex known as Cue1p. We have determined that a discrete region within Cue1p binds to the yeast ERAD E2 (Ubc7p) and is essential for ERAD in yeast. Interestingly, the CUE domain of Cue1p appears to be dispensible for function, despite the fact that the CUE domain of gp78 is required for its function. These studies in yeast complement and inform our studies in mammalian cells. They have also led to the identification of a discrete site of binding for Ubc7p on Cue1p, which we refer to as the Ubc7p Binding Region (U7BR). Studies are ongoing to further characterize this region at a biochemical and structural level. It is our hope that our work on Ubc7p and Cue1p will inform studies on gp78 and other mammalian RING finger ubiquitin ligases.